Rapid development of ultrasound contrast agents in the recent years has generated a number of different formulations, which are useful in ultrasound imaging of organs and tissue of human or animal body. These agents are designed to be used primarily as intravenous or intra-arterial injectables in conjunction with the use of medical echographic equipment which employs for example, B-mode image formation (based on the spatial distribution of backscatter tissue properties) or Doppler signal processing (based on Continuous Wave or pulsed Doppler processing of ultrasonic echoes to determine blood or liquid flow parameters).
A class of injectable formulations useful as ultrasound contrast agents includes suspensions of gas bubbles having a diameter of few microns dispersed in an aqueous medium.
Use of suspensions of gas bubbles in carrier liquid, as efficient ultrasound reflectors is well known in the art. The development of microbubble suspensions as echopharmaceuticals for enhancement of ultrasound imaging followed early observations that rapid intravenous injections of aqueous solutions can cause dissolved gases to come out of solution by forming bubbles. Due to their substantial difference in acoustic impedance relative to blood, these intravascular gas bubbles were found to be excellent reflectors of ultrasound. The injection of suspensions of gas bubbles in a carrier liquid into the blood stream of a living organism strongly reinforces ultrasonic echography imaging, thus enhancing the visualisation of internal organs. Since imaging of organs and deep seated tissues can be crucial in establishing medical diagnosis, a lot of effort has been devoted to the development of stable suspensions of highly concentrated gas bubbles which at the same time would be simple to prepare and administer, would contain a minimum of inactive species and would be capable of long storage and simple administration.
The simple dispersion of free gas bubbles in the aqueous medium is however of limited practical interest, since these bubbles are in general not stable enough to be useful as ultrasound contrast agents.
Interest has accordingly been shown in methods of stabilising gas bubbles for echography and other ultrasonic studies, for example using emulsifiers, oils, thickeners or sugars, or by entrapping or encapsulating the gas or a precursor thereof in a variety of systems. These stabilized gas bubbles are generally referred to in the art as “microvesicles”, and may be divided into two main categories.
A first category of stabilized bubbles or microvesicles is generally referred to in the art as “microbubbles” and includes aqueous suspensions in which the bubbles of gas are bounded at the gas/liquid interface by a very thin envelope (film) involving a stabilizing amphiphilic material disposed at the gas to liquid interface. Microbubbles suspensions are typically prepared by contacting powdered amphiphilic materials, e.g. freeze-dried preformed liposomes or freeze-dried or spray-dried phospholipid solutions, with air or other gas and then with an aqueous carrier, while agitating to generate a microbubble suspension which can then be administered, preferably shortly after its preparation.
Examples of aqueous suspension of gas microbubbles and preparation thereof are disclosed, for instance, in U.S. Pat. Nos. 5,271,928, 5,445,813, 5,413,774, 5,556,610, 5,597,549, 5,827,504 and WO 04/069284, which are here incorporated by reference in their entirety.
A second category of microvesicles is generally referred to in the art as “microballoons” or “microcapsules” and includes suspensions in which the bubbles of gas are surrounded by a solid material envelope of a lipid or of natural or synthetic polymers. Examples of microballoons and of the preparation thereof are disclosed, for instance, in U.S. Pat. Nos. 5,711,933 and 6,333,021, herein incorporated by reference in their entirety.
Microvesicles bearing an overall net charges are also known (see for instance International patent application WO 97/29783, herein incorporated by reference); the outer envelope of these microvesicles contains ionic compounds which are capable to confer the desired overall charge to the final microvesicle.
Further to these formulations of gas-filled microvesicles, interest has more recently been shown also towards modified formulations of gas-filled microvesicles, either for improving the diagnostic effect and/or for therapeutic purposes.
For instance, the microvesicles can be associated (e.g. by inclusion in its boundary envelope) with specific components (known as “targeting ligands”) which are capable to link to a determined target within a patient's body, e.g. to a specific pathogenic site. These formulations are generally known in the art as “targeted microvesicles”. Examples of targeted microvesicles, of targeting ligands and of the preparation thereof are disclosed for instance in International patent application WO 98/18051.
Another example of modified formulations are those where a therapeutic agent is associated with the microvesicle. When the formulation comprising the microvesicle reaches the pathogenic site, the drug can be advantageously released, e.g. by applying a controlled acoustic energy capable of disrupting the vesicle, thus locally releasing the therapeutic agent. This technique is generally known in the field as “ultrasound-mediated drug release”. Examples of microvesicles' formulations comprising a therapeutic agent are disclosed for instance in International patent application WO 94/28873.
Further developments in the field have brought to the preparation of assemblies wherein the microvesicle is associated with a second component, bearing a desired therapeutic agent or targeting compound.
For instance, WO 99/39738, discloses an assembly comprising a gas-filled microvesicle and a liquid-filled liposome associated therewith, where the liposome comprises a therapeutically active substance therein. The liposome is associated to the microvesicle by simple admixture with microvesicles or through a link between a conjugated pair, each of the microvesicle and liposome being provided with a component bearing one of the two the respective complementary moieties of said pair (e.g. biotin and avidin or streptavidin).
WO 03/015831 discloses a formulation comprising gas-filled microvesicles (“microspheres” in the application) associated to liposomes, referred to as microsphere-liposome composites. The liposomes of the composite may include a drug and/or a targeting moiety. The microvesicles and liposomes forming the composite are made from a same starting material; the composite is obtained preparing an aqueous solution comprising a mixture of lipids, introducing said solution in a sealed vial comprising the desired gas and finally agitating the solution. The so obtained composite is thus a simple mixture of microvesicles and liposomes of the same chemical nature. In particular, no specific chemical or physical interaction between microvesicles and liposomes is disclosed in said document.
Furthermore, International patent application WO 99/53963 discloses a combined preparation which comprises a first composition comprising gas-filled microvesicles dispersed in an aqueous medium and stabilized by a material and a second composition which is an oil-in-water emulsion comprising a material which stabilize the emulsion. The surface materials stabilizing the microvesicles and the dispersed oil phase have affinity for each other. In one embodiment, said affinity is obtained by using surface materials with opposite charges, so that they interact and bind electrostatically to each other. Alternatively, the association of the respective surface materials may comprises compounds capable of interaction through chemical or biological binding. The oil of the emulsion is a substance which is capable of generating a gas or vapor pressure in vivo and is referred to as the “diffusable component”. The association of droplets of said emulsified substance with the microvesicle is capable of determining a controllable growth of the dispersed gas phase in the microvesicle, through inward diffusion thereto of molecules of gas or vapour from said substance.